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Protein engineering on subtilisin E

L Zhu1, Y Ji

  • 1Department of Protein Engineering, Chinese Academy of Sciences, Beijing, China.

Chinese Journal of Biotechnology
|January 1, 1997
PubMed
Summary
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Protein engineering of subtilisin E using mutagenesis created four mutants with altered properties. Specific mutations enhanced oxidation resistance, thermal stability, and enzyme activity, though some reduced stability.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Enzyme Engineering

Background:

  • Subtilisin E is a widely used industrial enzyme.
  • Enhancing its stability and activity is crucial for various applications.
  • Protein engineering offers a pathway to modify enzyme properties.

Purpose of the Study:

  • To engineer subtilisin E variants with improved characteristics.
  • To investigate the impact of specific mutations on enzyme properties.
  • To characterize novel subtilisin E mutants.

Main Methods:

  • Site-directed and random mutagenesis of the subtilisin E gene.
  • Gene recombination in a shuttle vector and transformation into B. subtilis.
  • Purification and characterization of mutant enzymes, including stability and activity assays.

Related Experiment Videos

  • Isoelectric focusing-polyacrylamide gel electrophoresis (IEF-PAGE) to determine isoelectric points.

    • Main Results:

    • Four subtilisin E mutants were successfully generated and expressed.
    • M222A mutation conferred oxidation resistance.
    • N118S mutation enhanced thermal stability.
    • Q103R and D60N mutations increased specific activity but decreased thermal stability, with D60N causing significant instability.
    • Isoelectric points (pI) varied among mutants, ranging from 8.88 to 9.17.
    • Optimal pH for activity differed based on the substrate, with a broader range for casein.

    Conclusions:

    • Specific amino acid substitutions can fine-tune subtilisin E properties.
    • M222A and N118S mutations offer benefits in oxidation resistance and thermal stability, respectively.
    • While Q103R and D60N enhance activity, their impact on stability requires careful consideration for practical applications.